Effect of praziquantel treatment on pulmonary lesions of rats infected with Paragonimus iloktsuenensis

An experimental pathological study was performed to observe the effect of praziquantel treatment on the pulmonary lesions of the rat lung fluke, Paragonimus iloktsuenensis. The metacercariae were obtained from the freshwater crab, Sesarma dehaani, and 40 rats (wistar) were fed each with 10 metacercariae. On 20 rats praziquantel treatment (100mg/kg/day x 5 days) was done at 5 weeks after the infection while remaining 20 rats were kept untreated for use as controls. The drug-treated rats and the untreated ones were sacrificed 3, 7, 14, 21 or 28 days later for the observation of lung pathology. The rats infected with P. iloktsuenensis showed remarkable pulmonary changes; gross features of hemorrhagic and nodular worm capsules protruded on to the surface of the lung, and histologically local atelectasis, inflammatory cell infiltration, and egg granuloma around the worm capsules each containing one or two worms. Praziquantel treatment of the rats was shown to be highly effective in killing the worms and to lead them to degenerate, as early as in 3 days post-treatment. Almost all worms in the lung were dead and absorbed by the host cells in 21 days post-treatment, except a few living ones seen in a rat of 14-day post-treatment group. In most of the rats treated the pulmonary lesions showed the signs of resolution; regression of worm capsules with mummification of worms, decrease of inflammatory cell infiltration, improvement in the degree of atelectasis, and decreases in the frequency and size of the egg granuloma. From the results it is concluded that praziquantel is highly effective for the treatment of rat P. iloktsuenensis infection in the lung, not only by its direct killing effect of the worms but also due to the excellent resolution capacity of the pulmonary tissues.     

Evidence for metacercarial polymorphism in lung flukes, Paragonimus ohirai and Paragonimus iloktsuenensis

Cross breeding experiments between 2 species of lung fluke, Paragonimus ohirai and Paragonimus iloktsuenensis, were carried out using metacercarial characteristics as distinguishing markers. All metacercariae of F1 obtained were identical to those of P. ohirai. In the F2 and BF1 of F1 X P. iloktsuenensis, both the P. ohirai and P. iloktsuenensis types of metacercariae appeared. In the BF1 of F1 X P. ohirai, however, only the P. ohirai type metacercariae were produced. No intermediate type between the 2 species appeared. The results obtained demonstrate that the differences in the metacercariae, which were previously regarded as the most important characteristic for specific discrimination between these 2 flukes, are only a hereditary phenomenon within a single species. The metacercarial form, number of cyst layers, and body size seem to be controlled by a couple of alleles or very closely linked genes following simple Mendelian inheritance. Furthermore, we confirmed that reproduction in the lung flukes depends on cross-fertilization.     

Tegumental ultrastructures of Paragonimus iloktsuenensis according to the developmental stages

A scanning electron microscopic study was performed to observe the tegumental ultrastructures of Paragonimus iloktsuenensis according to its developmental stages. The metacercariae were obtained from the liver of the brackish water crab, Sesarma dehaani. Juvenile and adult P. iloktsuenensis were recovered from the experimental rats on 2, 4 and 8 weeks after infection. The findings were summarized as follows: 1. The excysted metacercariae were characteristically gourd-shape, with their whole body surface beset with numerous spade-shape spines. The large, type II sensory papillae (non-ciliated round swellings) were arranged along the rim of the oral and ventral suckers, 11-12 and 6-8 in numbers respectively. 2. Two-week old juvenile worms, recovered chiefly from the liver of the experimental rats, were slender in body shape, with their ventral sucker near the anterior one-third level. The distribution of tegumental spines was less dense than in the excysted metacercariae. The spines were with 1-2 pointed tips and 3-4 longitudinal splits. Numerous ciliated knob-like, type I papillae were observed in both sides of the oral sucker, and 6 large, type II papillae were arranged along the rim of the ventral sucker. 3. Four-week old worms, recovered from the thoracic cavity and/or lung parenchyme of the experimental rats, were thicker than wide in body configuration, and their ventral sucker was located near the anterior one-fourth level. The tegumental spines at ventral surface were grouped, each group with 3-5 aggregated ones. The type I and type II papillae (small-sized) were distributed chiefly around the rim of two suckers. 4. Adult (eight-week old) worms, recovered from the capsules in the lung parenchyme, were very stout, and covered densely with bearfoot-like spines. At dorsal surface, cobblestone-like cytoplasmic processes were well-developed, with many tegumental spines embedded in them. It was observed in this study that the tegument of P. iloktsuenesis continued to change and differentiate as the worms grew to be adults.     

Experimental infection of Paragonimus iloktsuenensis to albino rats, dogs and cats

This study was performed to observe the susceptibility of dogs and cats as definitive hosts of Paragonimus iloktsuenensis. The metacercariae of this fluke were obtained from Sesarma dehaani collected at a focus near the mouth of Sumjin river in November, 1986 and February, 1987. The larvae isolated from the crabs were introduced per os into 7 albino rats, 2 dogs and 3 cats. The adults were recovered from the experimental animals, and they were morphologically observed and measured. The results were as follows: 1. The recovery rate of adult worms at 42 days after infection was 53.3% from three albino rats, 21.0% from a dog and 12.7% from two cats. Most of the worms were recovered from the worm capsules in the lungs. 2. The size of worms recovered from albino rats, a dog, and cats 42 days after infection averaged 6.3 x 3.2 mm, 6.3 x 3.0 mm, or 6.2 x 3.5 mm, respectively. There were little differences in the morphology of worms by different experimental animals. 3. The size of eggs from a dog was 88.9 x 49.3 microns, and that from cats was 84.3 x 53.7 microns on average. Dogs and cats were good definitive hosts of P. iloktsuenensis. This fact suggests that human infection by this fluke may be possible if the metacercariae were ingested.     

Infection status of Sesarma dehaani collected from Sumjin river delta with the metacercariae of Paragonimus iloktsuenensis

This study was performed to observe the recent infection status of Sesarma dehaani with the metacercariae of P. iloktsuenensis in the well-known enzootic focus, Sumjin river delta. A total of 74 Sesarma dehaani were collected from a focus near the mouth of the Sumjin river in November, 1986 and February, 1987. The crabs were examined for P. iloktsuenensis metacercariae by the method of Seo and Kwak(1972). The metacercariae of P. iloktsuenensis were found in the liver of the crabs. Among the 74 crabs examined, 47(63.5%) were found infected with 1-102 metacercariae (18.2 per crab). The infection rate and metacercarial density increased as the size of the crab was increased. From the results, it is suggested that the life cycle of P. iloktsuenensis is actively maintained in the Sumjin river basin.     

Mortality in Syrian hamsters infected with Paragonimus kellicotti.

In an attempt to find a small animal model for paragonimiasis, Syrian hamsters were infected with between 1 and 16 metacercariae of Paragonimus kellicotti. A definitive mortality dose-response was observed with 90% of all hamsters given 3 or more parasites succumbing to the infection within 35 days. Hamsters demonstrated acute pleuritis, reactive mesothelial hyperplasia, subpleural accumulations of reactive and mature plasma cells, neovascularization, fibrohistiocytic thickening with and without giant cells, raised fibroconnective tissue lesions, and granulomatous inflammation with hemorrhage. Perivascular plasmacytic (lymphocytic) infiltrate, multifocal bronchopneumonia, and parenchymal necrotizing suppurative granulomatous inflammation, hemorrhagic pneumonia, and diffuse sprinkling of eosinophils, neutrophils, and intraalveolar macrophages also were observed. The response observed here may represent a new small animal mortality model useful in the search for new compounds to treat early trematode infections.     

Serum IgE levels in rats infected with Paragonimus westermani]

Paragonimus westermani is a common fluke in Korea. The present study aimed to determine serum total IgE and specific IgG levels in experimental paragonimiasis of rats. Each Wistar rat was inoculated orally with 20-25 metacercariae of P. westermani from Cambaroides similis. Before and after infection (1, 2, 3, 4, 6, 8 weeks) of P. westermani, the blood was collected from the retro-orbital venous plexus of rats and kept serum at -70 degrees C. Serum total IgE and specific IgG levels were determined by the capture and conventional enzyme-linked immunosorbent assay, respectively. The results were as follows; 1. Serum IgE values were increased to 0.18 +/- 0.042 at 2 weeks, 0.28 +/- 0.151 at 4 weeks and 0.43 +/- 0.055 at 8 weeks after infection. The absorbances of non-infected rats ranged 0.07 +/- 0.021-0.12 +/- 0.025. 2. Specific IgG values were slightly increased at 3 weeks (0.20 +/- 0.032) and gradually increased up to 8 weeks (0.31 +/- 0.067) after infection. The absorbances of non-infected rats ranged 0.11 +/- 0.035-0.18 +/- 0.019. The present results suggested that P. westermani could elevate serum IgE and specific IgG antibodies in Wistar rats which were not a good definitive host.     

Characterization and localization of Paragonimus westermani antigen stimulating antibody formation in both the infected cat and rat

Applicability of the adult Paragonimus westermani antigen for detection of anti-immature P. westermani antibodies in experimentally infected rats, a paratenic host of this lung fluke, was examined. The serum antibodies of the cats and rats infected with P. westermani metacercariae were detected by enzyme-linked immunosorbent assay (ELISA) with the adult-fluke antigen. The ELISA titers of serum samples of the rats infected with only immature flukes were as high as those of the cats infected with adult flukes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and the immunoblotting technique showed that a major protein band of 27,000 daltons was recognized in the sera of the infected cats and rats. Immunoperoxidase staining applied on the sectioned flukes provided evidence showing that the antigenic substance was located on the surface of the gut epithelium and in the luminal contents in both adult and immature flukes. The adult-fluke antigen containing the 27,000-dalton substance is applicable as a standard antigen for diagnosis of paragonimiasis westermani in not only definitive hosts but also in paratenic hosts.     

Reactivities of 4 murine coronavirus antigens with immunized or naturally infected rat sera by enzyme linked immunosorbent assay

Four murine coronavirus antigens, sialodacryoadenitis virus (SDAV) strain TG, Parker's rat coronavirus (PCV) strain 8190, mouse hepatitis virus (MHV) strains S and NuU, were examined for their reactivities to hyperimmunized and naturally infected rat sera by ELISA. With the immunized sera, SDAV and PCV antigens reacted best with respective homologous sera. MHV antigens reacted with all antisera, anti-SDAV, anti-PCV, and anti-MHV-S at approximately the same level, and MHV-S showed a slightly higher reactivity than MHV-NuU. The reactivities of the sera from various colonies to these antigens were in the order--from high to low--of SDAV, MHV-S, MHV-NuU, and PCV. None of sera negative for SDAV antigen reacted positively to the other antigens. Within the sera positive for SDAV, the positivities were in the order of MHV-S, MHV-NuU, and PCV. These results suggested that, although homologous antigens are best to detect SDAV or PCV infection by ELISA, MHV antigen can be used if highly cross-reactive viral strain is selected.     

Optimization of phage-immobilized ELISA for autoantibody profiling in human sera

Phage libraries displaying cDNA or random peptides have been used for profiling autoantibodies in cancer. The detection of autoantibodies in human sera using phages displaying specific epitopes is usually performed by phage-immobilized ELISAs which can detect specific antibodies without identification of whole antigens. However, these ELISAs can give feeble detection signals that are indistinguishable from background signals which are caused by human sera. To improve the usefulness of phage ELISA for human sera, the conditions for each step in phage ELISA were optimized. The antigenicity of phage antigens was maximal when using coating buffer of neutral pH. By using protein-free blocking buffer and pre-adsorbing human sera with phage host cell ER2738 extracts significantly decreased non-specific signals. Finally, when these conditions were applied to phage ELISA using K10P1, the values of the negative controls were concentrated near cutoff values, which made the assay more reliable. The optimized phage ELISA conditions described here would increase the efficacy of detection specific autoantibodies in human sera.     

The reactivity of antigens of Mycoplasma pulmonis derived from mice and rats to naturally infected rat sera

The reactivity of antigens of 4 mouse and 3 rat derived Mycoplasma pulmonis strains to 20 naturally infected rat sera was studied. The optical density values of the same serum by enzyme-linked immunosorbent assay using the 7 strains as the antigen revealed no marked difference among the strains. M. pulmonis antigens recognized by the antibodies were analyzed by the Western immunoblot method. The antigens with molecular weights of 92 K, 66 K, and 58 K were recognized in the 7 strains at a high frequency.     

Anti-interferon-gamma antibodies in sera from HIV infected patients

High serum levels of antibodies to interferon-gamma (IFN-gamma) have been found in patients infected with human immunodeficiency virus (HIV). A radioimmunoassay (RIA) with a recombinant IFN-gamma protein or an affinity purified IFN-gamma preparation as antigens, was developed to detect the specific anti-IFN-gamma antibodies. Reactivity of sera to IFN-gamma was confirmed by Western blot analysis. These antibodies, however, do not seem to recognize the active site of the molecule, since they do not neutralize the antiviral IFN-gamma activity in a biological assay. These results enforce the hypothesis of the role of autoimmunization during the course of the disease.     

Epitope mapping human heat shock protein 90 with sera from infected patients

Abstract Epitope mapping with sera from a range of infected patients showed that antibodies are commonly produced which cross-react with a number of epitopes on human heat shock protein 90 (HSP 90). Such autoreactive antibodies were particularly frequent in patients suffering from systemic candidiasis (9 patients), invasive aspergillosis (6 patients), ABPA (2 patients), a patient with aspergilloma and one with malaria. The patient with malaria recognised similar epitopes to those with invasive aspergillosis and candidiasis including the highly conserved epitope LKVIRKVIRK and an epitope NNLGTI which was otherwise only recognised by patients with candidiasis. Crossreacting antibodies to relatively few epitopes occurred in patients with Enterococcus faecalis and Corynebacterium jeikeium endocarditis. This was contrasted with the results from 6 patients with systemic lupus erythematosus (SLE) who were positive on immunoblot against fungal HSP 90. These did not react with the above epitopes but reacted with other areas within human HSP 90.     

Serodiagnosis of microbiosis in mice with a quantitative assay of immunoglobulins by a microcomputer-introduced ELISA system. 1. Anti-Sendai virus antibodies in naturally infected mouse sera

An enzyme-linked immunosorbent assay system combined with microcomputer data analysis was established as a quantitative assay method of immunoglobulins. The assay system was applied to measure IgG and IgM levels of anti-microbe antibodies in animals, especially mouse and rat. And now the measurement of IgG and IgM levels (ng/ml) of anti-Sendai virus (HVJ) antibodies in naturally infected mice is available. The assay system could improve serodiagnosis in the specificity and sensitivity and in the rapid treatment of many serum samples. The operation of this system was performed by a microcomputer, FM 8 connected Titertek Multiskan MC. The limited sensitivity of this assay for IgG and IgM was 10 ng/ml and 30 ng/ml, respectively. Ninety-one of serum samples were positive for IgG and/or IgM (45 samples for IgG and IgM, 44 samples for IgG, 2 samples for IgM) to Sendai virus in the tested 279 mouse sera, and serum titers were ranged from 1: 10 to 1: 12,800 in the IgG, and from 1: 20 to 1: 160 in the IgM. In these titers, serum IgG and IgM amounts were estimated to be 0.1 to 154 micrograms/ml and 0.5 to 4.8 micrograms/ml, respectively. Relationships of serum titers and antibody amounts were almost consisted, being judged like that approximately 10 micrograms/ml is 1: 400, 30 micrograms/ml is 1: 1,600 in IgG, and 2.4 micrograms/ml is 1: 80, 4 micrograms/ml is 1: 160 in IgM.     

Ultracentrifugal studies of sera from cattle vaccinated or naturally infected with Brucella abortus

In conformity with the findings of previous investigators, it was shown by density gradient ultracentrifugation that the antibodies in sera collected from calves shortly after vaccination with Brucella abortus, strain 19, were entirely or mainly rapidly-sedimenting. These macroglobulin (19S or IgM) antibodies showed complement-fixing as well as agglutinative activity with Br. abortus antigen. In later bleedings from the same vaccinated calves, antibodies with an intermediate sedimentation rate, (IgG), were present, as well as IgM. Sera from 15 of 22 non-vaccinated, relatively recent field cases of brucellosis appeared to contain only the IgG class of antibodies. In one herd, however, two cows with IgM only and five with both IgM and IgA were found; all seven of these cattle had been serologically negative before their introduction into this known infected herd a few months earlier. The agglutinative activity of sera from four cases of brucellosis of long standing and from eight cows, 4 to 13 years of age, that had been vaccinated as calves, was confined to the IgG fraction.